title: Purification of Protease from Pseudomonas thermaerum GW1 Isolated from Poultry Waste Site.
abstract: An extracellular protease was purified from Pseudomonas thermaerum GW1 a new strain identified by morphological, biochemical and 16S rDNA sequencing. It was isolated from soil of Poultry waste site at Ghazipur near Ghaziabad, Delhi. The strain produces extra cellular protease in the culture media that was maintained at 37°C, 140 rpm. The media was harvested for protease after 48 hrs of incubation at 37°C in basal media supplemented with 1% casein. We report 6.08 fold purification of enzyme following ammonium sulphate precipitation and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated to be approximately 43,000 daltons as shown by casein zymography studies. The optimum pH for the proteolytic activity was pH 8.0 and enzyme remained stable between pH 5 -11 at 60°C. Interestingly Mn(2+) (5mM) activated enzyme activity by 5 fold, while Cu(2+), Mg(2+)and Ca(2+) moderately activated enzyme activity, where as Zn(2+), Fe(2+) and Hg(2+) inhibited enzyme activity. The protease produced was stable in presence of 50 % (v/v) ethylacetate and acetone. Isopropanol, methanol and benzene increased protease activity by 2.7, 1.3 and 1.1 fold respectively but was inhibited in presence of glycerol and DMSO. This organic solvent-stable protease could be used as a biocatalyst for enzymatic peptide synthesis.
authors.affiliation: Department of Biotechnology, Jaypee Institute of Information Technology (Deemed University) A-10, sec 62, Noida, Uttar Pradesh, India.
authors.forename: Smriti
authors.initials: S
authors.lastname: Gaur
authors.forename: Sarita
authors.initials: S
authors.lastname: Agrahari
authors.forename: Neeraj
authors.initials: N
authors.lastname: Wadhwa